This invention relates generally to a fluorescence polarization assay useful in the detection and evaluation of soluble epoxide hydrolase (sEH) inhibitors. This invention also relates to novel fluorescent probes used in the fluorescence polarization assay, and methods of manufacturing such fluorescent probes.
Epoxide hydrolases are a group of enzymes ubiquitous in nature, detected in species ranging from plants to mammals. These enzymes are functionally related in that they all catalyze the addition of water to an epoxide, resulting in a diol. Epoxide hydrolases are important metabolizing enzymes in living systems. Epoxides are reactive species and once formed are capable of undergoing nucleophilic addition. Epoxides are frequently found as intermediates in the metabolic pathway of xenobiotics. Thus in the process of metabolism of xenobiotics, reactive species are formed which are capable of undergoing addition to biological nucleophiles. Epoxide hydrolases are therefore important enzymes for the detoxification of epoxides by conversion to their corresponding, non-reactive diols.
In mammals, several types of epoxide hydrolases have been characterized including soluble epoxide hydrolase (sEH), also referred to as cytosolic epoxide hydrolase, cholesterol epoxide hydrolase, LTA4 hydrolase, hepoxilin hydrolase, and microsomal epoxide hydrolase (Fretland and Omiecinski, Chemico-Biological Interactions, 129: 41-59 (2000)). Epoxide hydrolases have been found in all tissues examined in vertebrates including heart, kidney and liver (Vogel, et al., Eur J. Biochemistry, 126: 425-431 (1982); Schladt et al., Biochem. Pharmacol., 35: 3309-3316 (1986)). Epoxide hydrolases have also been detected in human blood components including lymphocytes (e.g. T-lymphocytes), monocytes, erythrocytes, platelets and plasma. In the blood, most of the sEH detected was present in lymphocytes (Seidegard et al., Cancer Research, 44: 3654-3660 (1984)).
The epoxide hydrolases differ in their specificity towards epoxide substrates. For example, sEH is selective for aliphatic epoxides such as epoxide fatty acids while microsomal epoxide hydrolase (mEH) is more selective for cyclic and arene oxides. The primary known physiological substrates of sEH are four regioisomeric cis epoxides of arachidonic acid known as epoxyeicosatrienoic acids or EETs. These are 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid. Also known to be substrates are epoxides of linoleic acid known as leukotoxin or isoleukotoxin. Both the EETs and the leukotoxins are generated by members of the cytochrome P450 monooxygenase family (Capdevila, et al., J. Lipid Res., 41: 163-181 (2000)).
The various EETs appear to function as chemical mediators that may act in both autocrine and paracrine roles. EETs appear to be able to function as endothelial derived hyperpolarizing factor (EDHF) due to their ability to cause hyperpolarization of the membranes of vascular smooth muscle cells with resultant vasodilation (Weintraub, et al., Circ. Res., 81: 258-267 (1997)). EDHF is synthesized from arachidonic acid by various cytochrome P450 enzymes in endothelial cells proximal to vascular smooth muscle (Quilley, et al., Brit. Pharm., 54: 1059 (1997)); Quilley and McGiff, TIPS, 21: 121-124 (2000)); Fleming and Busse, Nephrol. Dial. Transplant, 13: 2721-2723 (1998)). In the vascular smooth muscle cells EETs provoke signaling pathways involving ADP ribosylation of various protein substrates, leading to activation of BKCa2+ (big Ca2+ activated potassium channels). This results in hyperpolarization of membrane potential, inhibition of Ca2+ influx and relaxation (Li et al., Circ. Res., 85: 349-356 (1999)). Endothelium dependent vasodilation has been shown to be impaired in different forms of experimental hypertension as well as in human hypertension (Lind, et al., Blood Pressure, 9: 4-15 (2000)). Hence, it is likely that enhancement of EETs concentration would have a beneficial therapeutic effect in hypertensive patients where this plays a causative role. Examples of other conditions where enhanced vasodilation could play a positive role include angina, diabetes, stroke, ischemia, and pulmonary hypertension.
Other effects of EETs that may influence hypertension involve effects on kidney function. Levels of various EETs and their hydrolysis products, the DHETs, increase significantly both in the kidneys of spontaneously hypertensive rats (SHR) (Yu, et al., Circ. Res. 87: 992-998 (2000)) and in women suffering from pregnancy induced hypertension (Catella, et al., Proc. Natl. Acad. Sci. U.S.A., 87: 5893-5897 (1990)). In the rat model, both cytochrome P450 and sEH activities were found to increase (Yu et al., Molecular Pharmacology, 2000, 57, 1011-1020). Addition of a known sEH inhibitor was shown to decrease the blood pressure to normal levels. Finally, male soluble epoxide hydrolase null mice exhibited a phenotype characterized by lower blood pressure than their wild-type counterparts (Sinal, et al., J. Biol. Chem., 275: 40504-40510 (2000)).
An analogous effect on smooth muscle appears to operate in the lungs involving epithelial cells and airway smooth muscle relaxation (Dumoulin, et al., Am. J. Physiol., 275 (Lung Cell. Mol. Physiol. 19): L423-L431 (1998); Kiss, et al., Am. J. Resp. Crit. Care Med., 161: 1917-1923 (2000)). Hence, disease states where airways are overly constricted such as asthma, COPD, and bronchitis could benefit from enhanced EETs levels.
EETs, especially 11,12-EET, also have been shown to exhibit anti-inflammatory properties (Node, et al., Science, 285: 1276-1279 (1999); Campbell, TIPS, 21: 125-127 (2000); Zeldin and Liao, TIPS, 21: 127-128 (2000)). Node, et al. have demonstrated 11, 12-EET decreases expression of cytokine induced endothelial cell adhesion molecules, especially VCAM-1. They further showed that EETs prevent leukocyte adhesion to the vascular wall and that the mechanism responsible involves inhibition of NF-KB and IKB kinase.
In addition to the physiological effect of some substrates of sEH (EETs, mentioned above), some diols, i.e. DHETs, produced by sEH may have potent biological effects. For example, sEH metabolism of epoxides produced from linoleic acid (leukotoxin and isoleukotoxin) produces leukotoxin and isoleukotoxin diols (Greene, et al., Arch. Biochem. Biophys. 376(2): 420-432 (2000)). These diols were shown to be toxic to cultured rat alveolar epithelial cells, increasing intracellular calcium levels, increasing intercellular junction permeability and promoting loss of epithelial integrity (Moghaddam et al., Nature Medicine, 3: 562-566 (1997)). Therefore these diols could contribute to the etiology of diseases such as adult respiratory distress syndrome where lung leukotoxin levels have been shown to be elevated (Ishizaki, et al., Pulm. Pharm. and Therap., 12: 145-155 (1999)). Hammock, et al. have disclosed the treatment of inflammatory diseases, in particular adult respiratory distress syndrome and other acute inflammatory conditions mediated by lipid metabolites, by the administration of inhibitors of epoxide hydrolase (WO 98/06261; U.S. Pat. No. 5,955,496).
A number of classes of sEH inhibitors have been identified. Among these are chalcone oxide derivatives (Miyamoto, et al. Arch. Biochem. Biophys., 254: 203-213 (1987)) and various trans-3-phenylglycidols (Dietze, et al., Biochem. Pharm. 42: 1163-1175 (1991); Dietze, et al., Comp. Biochem. Physiol. B, 104: 309-314 (1993)).
More recently, Hammock et al. have disclosed certain biologically stable inhibitors of sEH for the treatment of inflammatory diseases, for use in affinity separations of epoxide hydrolases and in agricultural applications (U.S. Pat. No. 6,150,415). The Hammock ""415 patent also generally describes that the disclosed pharmacophores can be used to deliver a reactive functionality to the catalytic site, e.g., alkylating agents or Michael acceptors, and that these reactive functionalities can be used to deliver fluorescent or affinity labels to the enzyme active site for enzyme detection (col. 4, line 66 to col. 5, line 5). Certain urea and carbamate inhibitors of sEH have also been described in the literature (Morisseau et al., Proc. Natl. Acad. Sci., 96: 8849-8854 (1999); Argiriadi et al., J. Biol. Chem., 275 (20) 15265-15270 (2000); Nakagawa et al. Bioorg. Med. Chem., 8: 2663-2673 (2000)).
As outlined in the discussion above, inhibitors of sEH could be useful in the treatment of diseases either by preventing the degradation of sEH substrates that have beneficial effects or by preventing the formation of metabolites that have adverse effects. Therefore, in vitro screens to discover compounds that inhibit sEH are desirable as tools for discovering such inhibitors. Enzymatic assays for sEH activity and for inhibitors of sEH have been reported in the literature and include, for example, a cytosolic sEH enzyme EET assay, a recombinant sEH enzyme EET assay, and a cellular sEH enzyme trans-diphenylpropene oxide (tDPPO) assay. Tests for inhibitors involve adding test compound to a solution of sEH, incubating for a period of time, adding substrate, incubating, and monitoring the formation of diol.
Early sEH assays required preparation and use of a tritium-labeled substrate, a partitioning step and radiometric analysis (S. Gill et al., Analyt. Biochem., 131: 273-282 (1983)). Development of chromatographic assays, for example using HPLC analysis, avoided the use of radiolabeled substrate (R. N. Wixtrom and B. D. Hammock, in Biochemical Pharmacol. and Toxicol., D. Zakim and D. A. Vessey, Editors, Vol. 1: 1-93 (1985)). Each of these assays are time consuming and not amenable to high throughput. Also, in assays which have a partitioning step, followed by analysis of substrate and metabolite (either radiometric or chromatographic), varying extraction efficiencies can make accurate quantitation of substrate and metabolite and discrimination between inhibitors of similar potency difficult.
An improved spectrophotometric assay was described (Dietz et al., Analy. Biochem., 216: 176-187 (1994)), that used s-NEPC (4-nitrophenyl (2S,3S)-2,3-epoxy-3-phenylpropyl carbonate) as a substrate. Enzymatic hydrolysis of the s-NEPC quantitatively releases 4-nitrophenol which is subsequently monitored spectrophotometrically at 405 nm. This method has a very low signal to noise ratio due to the fact that 4-nitrophenol does not have a strong absorbance signal. In addition, s-NEPC is subject to auto hydrolysis. Both these factors make data analysis challenging. Although this assay is amenable to a 96-well plate format, the low signal to noise ratio makes this assay unfeasible for an ultra-high throughput screening approach.
In contrast to these known assays, the sEH fluorescence polarization assay of the present invention is a very sensitive and highly reproducible assay. This facilitates the determination of structure-activity relationships and the ranking of closely related test sEH inhibitors. It also has a very high signal to noise ratio, is not subject to auto hydrolysis since it is not an enzyme assay and is amenable to high throughput screening.
The present invention is directed to a fluorescence polarization assay for the detection and evaluation of sEH inhibitors that overcomes the aforementioned disadvantages of known assays.
A key feature of the fluorescence polarization assay of the present invention is the use of a novel fluorescent probe that binds to the active site of soluble epoxide hydrolase. This fluorescent probe, which constitutes another aspect of the present invention, is a compound having the following formula (I):
X-spacer-R1-Yxe2x80x83xe2x80x83(I) 
wherein X is the radical of compound that binds to the active site of soluble epoxide hydrolase, Y is a fluorescent label, xe2x80x9cspacerxe2x80x9d is a direct bond or is a C1-C16 alkylene group, a C2-C16 alkenylene group or a C2-C16 alkynylene group, wherein any of the available xe2x80x94CH2xe2x80x94 groups present in the C1-C16 alkylene group, C2-C16 alkenylene group or C2-C16 alkynylene group can optionally be replaced with O, S(O)p wherein p is 0 to 2, or N(R2), R1 is selected from the group consisting of O, S, xe2x80x94N(R2)C(O)xe2x80x94, xe2x80x94C(O)N(R2)xe2x80x94, xe2x80x94N(R2)C(S)xe2x80x94, xe2x80x94C(S)N(R2)xe2x80x94, xe2x80x94N(R2)C(S)NHxe2x80x94, xe2x80x94NHC(S)N(R2), xe2x80x94N(R2)C(O)NHxe2x80x94, xe2x80x94NHC(O)N(R2), xe2x80x94SO2NR2xe2x80x94, xe2x80x94NR2SO2xe2x80x94, xe2x80x94CH2N(R2)xe2x80x94, xe2x80x94N(R2)CH2xe2x80x94, xe2x80x94CH2Sxe2x80x94, xe2x80x94SCH2xe2x80x94, xe2x80x94C(O)CH2Sxe2x80x94, xe2x80x94SC(O)CH2xe2x80x94, 
xe2x80x94NHCH2CH2Sxe2x80x94, xe2x80x94SCH2CH2NHxe2x80x94, xe2x80x94NC(O)Oxe2x80x94, xe2x80x94ONC(O)xe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94OC(O)xe2x80x94, xe2x80x94NHxe2x80x94Nxe2x95x90C(R2)xe2x80x94, xe2x80x94C(R2)xe2x95x90Nxe2x80x94NHxe2x80x94, xe2x80x94NHCH(R2)xe2x80x94, or xe2x80x94CH(R2)NHxe2x80x94, and R2 is selected from H or C1-3alkyl.
The fluorescence polarization assay of the present invention generally comprises the following steps:
(a) determining the fluorescence polarization values of the free fluorescent probe and the fluorescent probe bound to soluble epoxide hydrolase to obtain a range of fluorescence polarization values and selecting a reference fluorescence polarization value falling within that range;
(b) mixing the fluorescent probe with soluble epoxide hydrolase in a buffered aqueous solution;
(c) mixing a test compound with the mixture obtained in step (b) and incubating the resulting mixture of fluorescent probe, soluble epoxide hydrolase and test compound;
(d) measuring the fluorescence polarization value of the incubated mixture obtained in step (c) to obtain a test fluorescence polarization value; and
(e) determining the difference between the test fluorescence polarization value and the reference fluorescence polarization value;
wherein the difference in fluorescence polarization values obtained in step (e) indicates whether the test compound inhibits soluble epoxide hydrolase.
The assay of the present invention is very sensitive and highly reproducible and can detect compounds that positively or negatively affect probe binding to the active site of soluble epoxide hydrolase by analyzing corresponding changes in fluorescence polarization, e.g., both competitive and allosteric inhibitors of sEH can be easily detected and evaluated. This assay is useful with respect to a variety of soluble epoxide hydrolases from different species and can also be used in high throughput screening procedures, e.g., efficiently screening a library of test compounds for soluble epoxide hydrolase inhibitory activity.